Lipotoxic endoplasmic reticulum (ER) stress during non-alcoholic fatty liver disease (NAFLD) can provoke hepatocyte death, inflammation and fibrosis, collectively known as non-alcoholic steatohepatitis (NASH). On the other hand, simple fatty liver does not rapidly progress into NASH, and this may be due to endogenous mechanisms that protect cells against ER stress. It is important to understand how NAFLD progression is attenuated, as this can lead to new preventive or therapeutic approaches against NASH. We have recently described a novel unfolded protein response (UPR) mechanism that is mediated by a stress-inducible protein Sestrin2. Sestrin2 is expressed upon obesity-induced ER stress and subsequently limits unfolded protein accumulation that facilitates NAFLD progression. Specifically, Sestrin2-mediated suppression of mTOR complex 1 (mTORC1) was shown to be critical for inhibiting protein translation and maintaining ER proteostasis during chronic phase of obesity-induced ER stress. Loss of Sestrin2 abolished this control and rendered hepatocytes hypersensitive to ER stress. Correspondingly, Sesn2-/- mice exhibited drastic acceleration of NAFLD progression upon obesity. Although this study has revealed that Sestrin2 controls ER homeostasis mainly through the regulation of mTORC1 and protein metabolism, we still do not have a clear molecular mechanism underlying Sestrin2-dependent regulation of mTORC1 and subsequent control of liver physiology. Thus, our main objective in this proposal is to determine the molecular mechanism of how Sestrin2 suppresses mTORC1 in the context of NAFLD. The rationale for the proposed research is that understanding how Sestrin2 supports liver homeostasis may lead to clinically-relevant attenuation of NAFLD progression through the development of Sestrin-like small molecule drugs that can mimic the effect of Sestrin2 on mTORC1. To approach this, we have performed tandem affinity purification (TAP)-mass spectrometry (MS) analyses that identified several Sestrin2-interacting proteins. Based on these results, we propose to pursue the following two aims: (1) Define the molecular mechanism of how Sestrin2 controls mTORC1 in liver; (2) Determine how Sestrin2 suppresses ER stress-associated liver pathologies. The proposed studies will enhance our understanding of the molecular network associated with pathological progression of NAFLD and provide a novel rationale for treatment and prevention of chronic liver diseases, harnessing the beneficial activities of Sestrin2 and other Sestrins. As this study may also provide conceptually new perspectives in understanding cellular adaptation to ER stress, the new knowledge gained from this research will also help bring out innovative changes in comprehending and treating a wide range of obesity- and ER stress-associated diseases in liver and other tissues beyond the scope of NAFLD.